Fabrication of vascularized tissue using microfabricated two-dimensional molds

ABSTRACT

A method and materials to create complex vascularized living tissue in three dimensions from a two-dimension microfabricated mold has been developed. The method involved creating a two dimensional surface having a branching structure etched into the surface. The pattern begins with one or more large channels which serially branch into a large array of channels as small as individual capillaries, then converge to one or more large channels. The etched surface serves a template within a mold formed with the etched surface for the circulation of an individual tissue or organ. Living vascular cells are then seeded onto the mold, where they form living vascular channels based on the pattern etched in the mold. Once formed and sustained by their own matrix, the top of the mold is removed. The organ or tissue specific cells are then added to the etched surface, where they attach and proliferate to form a thin, vascularized sheet of tissue. The tissue can then be gently lifted from the mold using techniques such as fluid flow and other supporting material, as necessary. The tissue can then be systematically folded and compacted into a three-dimensional vascularized structure. This structure can then be implanted into animals or patients by directly connecting the blood vessels to flow into and out of the device. Immediate perfusion of oxygenated blood occurs, which allows survival and function of the entire living mass.

This application claims priority ot U.S. Ser. No. 60/131,930 filed Apr.30, 1999, and U.S. Ser. No. 60/165,329 filed Nov. 12, 1999.

The United States government has certain rights in this invention byvirtue of grant number DAMD 17-99-2-9001 from the Department of Defense.

BACKGROUND OF THE INVENTION

The present invention generally relates to the fields of organtransplantation and reconstructive surgery, and to the new field ofTissue Engineering. It more specifically is a new method and materialsfor generating tissues requiring a blood vessel supply and other complexcomponents such as a nerve supply, drainage system and lymphatic system.

Organ transplantation, as currently practices, has become a majorlifesaving therapy for patients afflicted with disease which destroyvital organs including the heart, liver, lungs, kidney and intestine.However, the shortage of organs needed for transplantation has becomecritical and continues to worsen. Likewise, every major field ofreconstructive surgery reaches the same barrier of tissue shortage.Orthopedic surgery, vascular surgery, cardiac surgery, general surgery,neurosurgery, and the others all share this fundamental problem.Therefore, countless patients suffer as a result.

Over the last twelve years, the new field of tissue engineering hasarisen to meet this need. The field brings the expertise of physicians,life scientists and engineers together to solve problems of generatingnew tissues for transplantation and surgical reconstruction. The initialapproaches to this problem were described in the 1980's. Yannas andBurke (Bell, et al., Science 221,1052 (1981); Burke, et al., Ann Surg194, 413 (1981)(described methods to generate new tissues in vivo byimplanting non-living materials such as modified collagens which areseeded with cells to promote guided regeneration of tissue such as skin.Vacanti and Langer (Langer and Vacanti Science 260, 920 (1993); Vacanti,et al., Materials Research Society 252,367 (1992)) described syntheticfibrous matrices to which tissue specific cells were added in vitro. Thematrices are highly porous and allow mass transfer to the cells in vitroand after implantation in vivo. After implantation, new blood vesselsgrow into the devices to generate a new vascularized tissue. However,the relatively long time course for angiogenesis limits the size of thenewly formed tissue.

The field of Tissue Engineering is now maturing and undergoing explosivegrowth. See, for example, Vacanti and Langer, Lancet 354, 32 (1999);Langer and Vacanti Science 260, 920 (1993); Rennie, J. ed. Specialreport: The promise of tissue engineering. Scientific American 280, 37(1999); and Lysaght, et al., Tissue Eng 4, 231 (1998). Virtually everytissue and organ of the body has been studied. Many tissue-engineeringtechnologies are becoming available for human use. See, Lysaght, et al.Tissue Eng 4, 231 (1998); Bell, et al., Science 221,1052 (1981); Burke,et al., Ann Surg 194, 413 (1981); Compton, et al., LaboratoryInvestigation 60, 600 (1989); Parenteau, et al., Journal of CellularBiochemistry 45, 24 (1991); Parenteau, et al., Biotechnology andBioengineering 52, 3 (1996); Purdue, et al., J. Burn Care Rehab 18, 52(1997); Hansbrough and Franco, Clinical Plastic Surg 25, 407 (1998);Vacanti, et al., Materials Research Society 252,367 (1992).

Over time, several techniques to engineer new living tissue have beenstudied. Technologies include the use of growth factors to stimulatewound repair and regeneration, techniques of guided tissue regenerationusing non-living matrices to guide new tissue development, celltransplantation, and cell transplantation on matrices. More recently,new understanding in stem cell biology has led to studies of populationsof primordial cells, stem cells, or embryonic stem cells to use intissue engineering approaches.

To date, all approaches in tissue engineering have relied on thein-growth of blood vessels into tissue-engineered devices to achievepermanent vascularization. This strategy has worked well for manytissues. However, it falls short for thick, complex tissues such aslarge vital organs, including liver, kidney, and heart. Techniques usingthree-dimensional printing technology to achieve ordered arrays ofchannels have been described to begin to solve this problem. See, forexample, Griffith, et al., Ann NY Acad Sci 831, 382 (1997); Langer andVacanti JP Sci Am 280, 62 (1999).

In parallel to these advances, the rapidly emerging field ofMicroElectroMechanical Systems (MEMS) has penetrated a wide array ofapplications, in areas as diverse as automotives, inertial guidance andnavigation, microoptics, chemical and biological sensing, and, mostrecently, biomedical engineering, Langer and Vacanti Sci Am 280, 62(1999); McWhorter, et al. “Micromachining and Trends for theTwenty-First Century”, in Handbook of Microlithography, Micromachiningand Microfabrication, ed. P. Rai-Choudhury, (Bellingham, Wash.: SPIEPress, 1997). Microfabrication methods for MEMS represent an extensionof semiconductor wafer process technology originally developed for theintegrated circuit (IC) industry. Control of features down to thesubmicron level is routinely achieved in IC processing of electricalcircuit elements; MEMS technology translates this level of control intomechanical structures at length scales stretching from less than 1micron to greater than 1 cm. Standard bulk micromachining enablespatterns of arbitrary geometry to be imprinted into wafers using aseries of subtractive etching methods. Three-dimensional structures canbe realized by superposition of these process steps using precisealignment techniques. Several groups (Kourepenis, et al., “Performanceof MEMS Inertial Sensors,” Proc. AIAA GN&C Conference, Boston, Mass.,1998; Griffith, et al., Annals of Biomed. Eng., 26 (1998); Folch, etal., Biotechnology Progress, 14, 388 (1998)) have used these highlyprecise silicon arrays to control cell behavior and study geneexpression and cell surface interactions. However, this approach isessentially a two-dimensional technology and it has not been apparentthat it might be adapted to the generation of thick, three-dimensionaltissues.

PCT US96/09344 by Massachusetts Institute of Technology describe athree-dimensional printing process, a form of solid free formfabrication, which builds three-dimensional objects as a series oflayers. This process uses polymer powders in layers bound by polymerbinders whose geometry is dictated by computer assisted design andmanufacture. This technique allows defined internal architectures whichcould include branching arrays of channels mimicking a vascular supply.However, this technique is limited by the characteristics and chemistryof the particular polymers. Also, it severely limits the types of tissueto be fabricated. Polymer walls do not allow the plasma exchange that isneeded at the alveolar capillary wall of the lung.

The object of the present invention is to provide a method and materialsfor creating complex, living vascularized tissues for organ and tissuereplacement, especially complex and/or thick structures, such as livertissue.

SUMMARY OF THE INVENTION

A method and materials to create complex vascularized living tissue inthree dimensions from a two-dimension microfabricated mold has beendeveloped. The method involved creating a two dimensional surface havinga branching structure etched into the surface. The pattern begins withone or more large channels which serially branch into a large array ofcharmels as small as individual capillaries, then converge to one ormore large channels. The etched surface serves a template within a moldformed with the etched surface for the circulation of an individualtissue or organ. Living vascular cells are then seeded onto the mold,where they form living vascular channels based on the pattern etched inthe mold. Once formed and sustained by their own matrix, the top of themold is removed. The organ or tissue specific cells are then added tothe etched surface, where they attach and proliferate to form a thin,vascularized sheet of tissue. The tissue can then be gently lifted fromthe mold using techniques such as fluid flow and other supportingmaterial, as necessary. The tissue can then be systematically folded andcompacted into a three-dimensional vascularized structure. Thisstructure can then be implanted into animals or patients by directlyconnecting the blood vessels to flow into and out of the device.Immediate perfusion of oxygenated blood occurs, which allows survivaland function of the entire living mass.

The design of the branching channels can be constructed by a number ofmeans, such as fractal mathematics which can be converted by computersinto two-dimensional arrays of branches and then etched onto wafers.Also, computers can model from live or preserved organ or tissuespecimens three dimensional vascular channels, convert totwo-dimensional patterns and then help in the reconversion to athree-dimensional living vascularized structure. Techniques forproducing the molds include techniques for fabrication of computer chipsand microfabrication technologies. Other technologies include lasertechniques. The two-dimensional surface of the mold can also be variedto aid in the folding and compacting process. For example, the surfacecan be changed from planar to folded accordian like. It can be stackedinto multiple coverging plates. It could be curvilinear or have multipleprojections.

Different types of tissue, or multiple layers of the same type oftissue, can be placed adjacent to each other prior to folding andcompacting, to create more complex or larger structures. For example, atubular system can be layered onto a vascular system to fabricateglomerular tissue and collecting tubules for kidneys. Bile duct tubescan be onlaid over vascularized liver or hepatocyte tissue, to generatea bile duct drainage system. Alveolar or airway tissue can be placed onlung capillaries to make new lung tissue. Nerves or lymphatics can beadded using variations of these same general techniques. Thetwo-dimensional surface of the mold can also be varied to aid in thefolding and compacting process. For example, the surface can be changedfrom planar to folded accordian like. It can be stacked into multiplecoverging plates. It could be curvilinear or have multiple projections.

Examples of tissues and organs which can be fabricated using thesemethods include, but are not restricted to, organs currentlytransplanted such as heart, liver, lung, kidney and intestine. Othertissues such as muscle, bone and breast tissue could also be engineered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a schematic of the process for making a branching patternono a silicon substrate. FIG. 1 b is a more detailed schematic showingthe process to make a more complex structure, with channels of varyingdepths.

FIG. 2 a is a schematic of an etched surface showing a branchingstructure which branches out from a single inlet and then converges backinto a single outlet. FIGS. 2 b, c, and d are schematics of across-sectional view of different etched channels in the surface of FIG.2 a.

FIGS. 3 a-g are schematics of multiple tissue layers assembled to formthree dimensional structures.

FIGS. 4 a, b and c are schematics of the process for making a tissuelayer.

FIG. 5 is a schematic of an assembled complex tissue or organ formed bythe process of FIGS. 4 a-c.

FIG. 6 shows how the organ of FIG. 5 can be connected to a fluid byanastomosis of the inlet and outlet.

FIG. 7 is a schematic of the pattern etched using an inductively-coupled(IPC) system.

FIG. 8 is a schematic of the process for fabricating U-shaped trenchesin silicon wafers.

FIG. 9 a shows the vascular branching network pattern used for siliconand pyrex wafer micromachining (FIG. 9 a), FIG. 9 b shows the opticalmicrograph or portion of the capillary network etched into the siliconwafer using the process shown in FIG. 7, and FIG. 9 c is a scanningelectron micrograph of the anisotrophic etching process used to formangled sidewall trenches.

DETAILED DESCRIPTION OF THE INVENTION

Due to the difficulties of the prior art, an approach to provide acomplete vascular system to the engineered structure before implantationwas developed using microfabrication techniques such as threedimensional printing to provide an ordered array of branching channelsin a substrate formed of a material such as silicon or a biocompatiblepolymer, which are then seeded with cells. A complete branching vascularcirculation is made in two dimensions on the surface of silicon usingmicrofabrication. The vascular circulation is then lifted from thesilicon mold and folded or rolled into a compact three dimensionalstructure.

Microfabrication technology has been used in important studies in celland developmental biology to understand complex biologic signalingevents occurring at the cell membrane-surface interface, as described,for example, by Kane, et al., Biomaterials 20, 2363 (1999). It has alsobeen used in tissue engineering to guide cell behavior and the formationof small units of tissue, as described by Griffith, et al., Annals ofBiomed. Eng., 26 (1998). As demonstrated by Example 1, a coherentstructure over a broad range of scale has now been made whichdemonstrates the efficacy of this method for tissue engineering forconstruction of complex and/or thick structures such as liver. Thedevice constructed in the example includes channels that begin as asingle inlet channel with a diameter of 500 microns, branch through fourgenerations following a geometric scaling law which halves the channelwidth for each successive generation, form an array of capillarychannels 10 microns in diameter, and then sequentially branch back to asingle outflow vein. Living endothelial cells seeded into these channelsand provided with flow of appropriate nutrients and gases will line thechannels to form blood vessels. In Example 2, it has been demonstratedthat cells seeded onto surfaces of silicon and pyrex will lay downmatrix and form sheets of tissue of the cell type of origin, eitherhepatic or endothelial. These sheets can be peeled from the surface andformed into three dimensional units of tissue. In effect, the wafer ofsilicon or pyrex has acted as a mold for the formation of tissue.

These examples demonstrate that microfabrication technology can beadapted to suit the needs of forming living tissue. The power of thetechnique lies in its control of form over extremely small distances.The resolution is on the order of 0.1 microns from point to point. Thislevel of precision adds new levels of control in the ability to designand guide new tissue formation. For instance, surfaces can be imprintedwith submicron grooves or scallops, and corners can be made rounded,angled or sharp with this same level of submicron precision. Geometriccontrol at this scale can have a powerful impact on cell adhesionthrough mechanisms such as contact guidance, as described by Den Braber,et al. J Biomed. Mater. Res. 40, 291 (1998). This technology overcomesthe problems with the prior art which was limited to very thinstructures because designs were constrained to the surface of thesilicon wafers. Using the wafer as a temporary mold, then lifting thetissue from it and folding the tissue into three-dimensional space,overcomes this limitation.

As described herein, complex tissues are formed by laminating layers ofthin vascularized tissues to form thicker tissue structures or morecomplex organ equivalents. The thin vascularized tissue layers areformed by:

(1) designing a mold having a complex pattern of channels formed into atleast one surface into which cells can be seeded;

(2) seeding vascular (endothelial) cells into the channels;

(3) culturing the vascular cells under conditions until they formvasculature;

(4) seeding other type(s) of cells onto or into the mold so that atissue is formed incorporating the vasculature;

(5) removing the vascularized tissue layer; and

(6) assembling multiple layers of the vascularized tissue layers untilthe desired complex structure (or organ equivalent) is formed.

This structure can then be implanted and the vasculature, if properlydesigned, anastomized into the existing vasculature to provide animmediate blood supply for the implanted organ equivalent.

Molds for Manufacturing Thin Vascularized Tissue Layers

Materials for Forming Molds

Fabrication of the wafer molds begins by selection of an appropriatesubstrate. Any of a variety of materials can be used to form thesurfaces on which the branching structures can be molded or etched.These include “inert” materials such as silicone, polymers such aspolyethylene vinyl acetate, polycarbonate, and polypropylene, andmaterials such as a ceramic or material such as hydroxyapatite. Inparticular, the mold can be constructed from metals, ceramics,semiconductors, organics, polymers, and composites. Representativemetals and semiconductors include pharmaceutical grade stainless steel,gold, titanium, nickel, iron, gold, tin, chromium, copper, alloys ofthese or other metals, silicon, silicon dioxide.

Typically, micromachining is performed on standard bulk single crystalsilicon wafers of a diameter ranging between 50 and 300 millimeters, andof thickness ranging between 200 and 1200 microns. These wafers can beobtained from a large number of vendors of standard semiconductorsmaterial, and are sawn and polished to provide precise dimensions,uniform crystallographic orientation, and highly polished, opticallyflat surfaces. Wafers made from pyrex borosilicate or other glasses canalso be procured and inserted into micromachining processes, withalternative processes used to etch the glassy materials.

The choice of a substrate material is guided by many considerations,including the requirements placed on the fabrication process by thedesired mold dimensions, the desired size of the ultimate template, andthe surface properties of the wafer and their interaction with thevarious cell types, extracellular matrix (“ECM”) and polymeric backbone.Cost may also be a consideration, depending upon the overall sizerequirements of the tissue mold.

Unless otherwise specified, the term “polymer” includes polymers andmonomers which can be polymerized or adhered to form an integral unit.The polymer can be non-biodegradable or biodegradable, typically viahydrolysis or enzymatic cleavage, although biodegradable matrices arenot typically preferred since the molds are not implanted and arepreferably reusable. Non-polymeric materials which can also be usedinclude organic and inorganic materials such as hydoxyapatite, calciumcarbonate, which are solidified by application of adhesive rather thansolvent. In a preferred embodiment, polymers are selected based on theability of the polymer to elicit the appropriate biological responsefrom cells, for example, attachment, migration, proliferation and geneexpression. As noted above, many non-biodegradable plastics are knownwhich are currently in use in cell culture, including polystyrene,polycarbonate, polypropylene, polyvinylacetate, as well as biodegradablepolymers such as polyhydroxy acids and polyhydroxyalkanoates.Photopolymerizable, biocompatible water-soluble polymers includepolyethylene glycol tetraacrylate (Ms 18,500) which can bephotopolymerized with an argon laser under biologically compatibleconditions using an initiator such as triethanolamine,N-vinylpyrrolidone, and eosin Y. Other suitable polymers can be obtainedby reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989).

Solvents for most of the thermoplastic polymers are known, for example,methylene chloride or other organic solvents. Organic and aqueoussolvents for protein and polysaccharide polymers are also known. Thebinder can be the same material as is used in conventional powderprocessing methods or may be designed to ultimately yield the samebinder through chemical or physical changes that occur as a result ofheating, photopolymerization, or catalysis.

These can be coated with a material enhancing cell adhesion. In someembodiments, attachment of the cells to the polymer is enhanced bycoating the substrate with compounds such as basement membranecomponents, agar, agarose, gelatin, gum arabic, collagens types I, II,III, IV, and V, fibronectin, laminin, glycosaminoglycans, mixturesthereof, and other materials known to those skilled in the art of cellculture.

Properties of the mold surface can also be manipulated through theinclusion of materials on or in the mold material which alters porosity,cell attachment (for example, by altering the surface charge orstructure), flexibility or rigidity (which may be desirable tofacilitate removal of tissue constructs).

Methods for Designing Mold Surfaces

Once the substrate material has been selected, the process sequence formold generation must be defined. The geometry of the mold, in particularthe number of different feature depths required, is the major factordetermining the specific process sequence. The simplest case is that ofa single depth dimension for the mold. Specifically, for a siliconsubstrate, the process sequence (shown in FIG. 1 a) is as follows:First, the silicon wafer is cleaned, and a layer of photosensitivematerial is applied to the surface. Typically, the layer is spun on at ahigh revolution rate to obtain a coating of uniform thickness. Thephotoresist is baked, and the wafer is then exposed to ultraviolet orother short-wavelength light though a semi-transparent mask. This stepcan be accomplished using any one of several masking techniques,depending on the desired image resolution. The resist is then developedin an appropriate developer chemistry, and the wafer is then hard-bakedto remove excess solvent from the resist. Once the lithographic processhas been completed, the wafer can be etched in a plasma reactor usingone of several possible chemistries. Etching serves to transfer thetwo-dimensional pattern into the third dimension: a specified depth intothe wafer. Plasma parameters are determined by the desired shape of theresulting trench (semi-circular, straight-walled profile, angledsidewall), as well as by the selectivity of the etchant for silicon overthe masking photoresist. Once the etching has been completed, thephotoresist can be removed and the wafer prepared for use in the tissuemolding process.

Glass and polymeric wafer molds can be fabricated using a similarsequence, but the actual process may be modified by the addition of anintervening masking layer, since etchants for these materials may attackphotoresist as well. Such intervening materials simply function totransfer the pattern from the photoresist to interlayer and then on tothe wafer below. For silicon etched in one of several wet chemistries,an intervening layer may also be necessary.

Increased flexibility in the geometry of wafer mold may be obtained byinserting additional cycles of masking and etching, as shown in FIG. 1b. Here, a second step in which a masking layer has been applied andopen areas etched is shown. This modification provides the opportunityto machine channels of varying depths into the wafer mold. For vascularbranches with different diameters, this increased flexibility becomesvery important. The techniques may be extended to provide as manyadditional layers and different depths as is desired.

The mold surface is configured as required to create tissue sectionshaving the desired vascular or other tubular structures. In the casewhere a naturally occurring structure is mimicked, channels are etchedin the same two-dimensional pattern that occurs in the natural tissue. Adifference is that the natural tissue will not be limited to atwo-dimensional structure, but instead would include vasculature thatextends three dimensionally. This differs from the thin tissue or organsections created as described herein.

The design of the branching channels can be constructed by a number ofmeans, such as fractal mathematics which can be converted by computersinto two-dimensional arrays of branches and then etched onto wafers.Also, computers can model from live or preserved organ or tissuespecimensthree dimensional vascular channels, convert to two-dimensionalpatterns and then help in the reconversion to a three-dimensional livingvascularized structure. CAD-CAM type software programs would typicallybe most useful for design of these structures.

Methods for Configuring the Molds

The selection of the material which is used as the support determineshow the surface of the mold is configured to form the branchingstructure. Materials can be configured by molding, particularly in thecase of polymers, etching using techniques such as lasers, plasmaetching, or chemical etching, photolithography, or solid free formtechniques including three dimensional printing (3DP), stereolithography(SLA), selective laser sintering (SLS), ballistic particle manufacturing(BPM) and fusion deposition modeling (FDM), micromachining, orcombinations thereof.

Conventional Polymer Processing

Polymers can be configured using standard techniques such as solventcasting or extrusion molding into a pre-fabricated mold, shaped usingone of the solid free form techniques or configured after shaping, usingchemical etching, micromachining, lasers, or other methods describedherein. These methods can also be used to form the molds from materialsother than polymers.

Micromachining and Chemical Processing of Silicon Materials

In one embodiment, the mold devices are made by microfabricationprocesses, by creating small mechanical structures in silicon, metal,polymer, and other materials. These microfabrication processes are basedon well-established methods used to make integrated circuits and othermicroelectronic devices, augmented by additional methods developed byworkers in the field of micromachining.

Microfabrication processes that may be used in making the moldsdisclosed herein include lithography; etching techniques, such as wetchemical, dry, and photoresist removal; thermal oxidation of silicon;electroplating and electroless plating; diffusion processes, such asboron, phosphorus, arsenic, and antimony diffusion; ion implantation;film deposition, such as evaporation (filament, electron beam, flash,and shadowing and step coverage), sputtering, chemical vapor deposition(CVD), epitaxy (vapor phase, liquid phase, and molecular beam),electroplating, screen printing, and lamination. See generally Jaeger,Introduction to Microelectronic Fabrication (Addison-Wesley PublishingCo., Reading Mass. 1988); Runyan, et al., Semiconductor IntegratedCircuit Processing Technology (Addison-Wesley Publishing Co., ReadingMass. 1990); Proceedings of the IEEE Micro Electro Mechanical SystemsConference 1987-1998; Rai-Choudhury, ed., Handbook of Microlithography,Micromachining & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997). The following methods are preferred for makingmolds.

Electrolytic anodization of silicon in aqueous hydrofluoric acid,potentially in combination with light, can be used to etch channels intothe silicon. By varying the doping concentration of the silicon wafer tobe etched, the electrolytic potential during etching, the incident lightintensity, and the electrolyte concentration, control over the ultimatepore structure can be achieved.

This process uses deep plasma etching of silicon to create molds withdiameters on the order of 0.1 μm or larger. Needles are patterneddirectly using photolithography, rather than indirectly by controllingthe voltage (as in electrochemical etching), thus providing greatercontrol over the final mold geometry.

In this process, an appropriate masking material (e.g., metal) isdeposited onto a silicon wafer substrate and patterned into dots havingthe diameter of the desired molds. The wafer is then subjected to acarefully controlled plasma based on fluorine/oxygen chemistries to etchvery deep, high aspect ratio trenches into the silicon. See, e.g.,Jansen, et al., “The Black Silicon Method IV: The Fabrication ofThree-Dimensional Structures in Silicon with High Aspect Ratios forScanning Probe Microscopy and Other Applications,” IEEE Proceedings ofMicro Electro Mechanical Systems Conference, pp. 88-93 (1995).

In this process, a metal layer is first evaporated onto a planarsubstrate. A layer of photoresist is then deposited onto the metal toform a patterned mold which leaves an exposed-metal region in the shapeof needles. By electroplating onto the exposed regions of the metal seedlayer, the mold bounded by photoresist can be filled with electroplatedmaterial. Finally, the substrate and photoresist mold are removed,leaving the finished mold array. The molds produced by this processgenerally have diameters on the order of 1 μm or larger. See, e.g.,Frazier, et al., “Two dimensional metallic microelectrode arrays forextracellular stimulation and recording of neurons”, IEEE Proceedings ofthe Micro Electro Mechanical Systems Conference, pp. 195-200 (1993).

Another method for forming molds made of silicon or other materials isto use microfabrication techniques to make a mold form, transferringthat mold form to other materials using standard mold transfertechniques, such as embossing or injection molding, and reproducing theshape of the original mold form using the newly-created mold to yieldthe final molds. Alternatively, the creation of the mold form could beskipped and the mold could be microfabricated directly, which could thenbe used to create the final molds.

Another method of forming solid silicon molds is by using epitaxialgrowth on silicon substrates, as is utilized by Containerless Research,Inc. (Evanston, Ill., USA) for its products.

The size distribution of the etched porous structure is highly dependenton several variables, including doping kind and illumination conditions,as detailed in Lehmann, “Porous Silicon—A New Material for MEMS”, IEEEProceedings of the Micro Electro Mechanical Systems Conference, pp. 1-6(1996). Porous polymer molds can be formed, for example, by micromoldinga polymer containing a volatilizable or leachable material, such as avolatile salt, dispersed in the polymer, and then volatilizing orleaching the dispersed material, leaving a porous polymer matrix in theshape of the mold. Hollow molds can be fabricated, for example, usingcombinations of dry etching processes (Laermer, et al., “Bosch DeepSilicon Etching: Improving Uniformity and Etch Rate for Advanced MEMSApplications,” Micro Electro Mechanical Systems, Orlando, Fla., USA,(Jan. 17-21, 1999); Despont et al., “High-Aspect-Ratio, Ultrathick,Negative-Tone Near-UV Photoresist for MEMS”, Proc. of IEEE 10^(th)Annual International Workshop on MEMS, Nagoya, Japan, pp. 518-522 (Jan.26-30, 1997)); micromold creation in lithographically-defined polymersand selective sidewall electroplating; or direct micromolding techniquesusing epoxy mold transfers.

A chromium mask can be substituted for the solid molds using a siliconnitride layer covered with chromium. Solid molds are then etched, thechromium is stripped, and the silicon is oxidized. The silicon nitridelayer will prevent oxidation. The silicon nitride is then stripped,leaving exposed silicon and oxide-covered silicon everywhere else. Theneedle is then exposed to an ICP plasma which selectively etches thesilicon in a highly anisotropic manner to form the interior hole of theneedle. A second method uses solid silicon as ‘forms’ around which theactual needle structures are deposited. After deposition, the forms areetched away, yielding the hollow structures. Silica needles or metalneedles can be formed using different methods. The wafers are thenoxidized to a controlled thickness, the silicon nitride is then strippedand the silicon core selectively etched away (e.g., in a wet alkalinesolution) to form a hollow silica mold.

In another embodiment, deep reactive ion etching is combined with amodified black silicon process in a conventional reactive ion etcher.First, designs are patterned through photoresist into SiO₂, such as on asilicon wafer. Then the silicon can be etched using deep reactive ionetching (DRIE) in an inductively coupled plasma (ICP) reactor to etchdeep vertical holes or channels. The photoresist is then removed. Next,a second photolithography step patterns the remaining SiO₂ layer. Thephotoresist is then removed and the silicon wafer again deep siliconetched completely through the wafer in the regions not covered withSiO₂). This process can be varied as follows. After the wafer ispatterned, the photoresist and SiO₂ layers are replaced with conformalDC sputtered chromium. The second ICP etch is replaced with a SF₆/O₂plasma etch in a reactive ion etcher (RIE), which results in positivelysloping outer sidewalls. Henry, et al., “Micromachined Needles for theTransdermal Delivery of Drugs,” Micro Electro Mechanical Systems,Heidelberg, Germany, pp. 494-498 (Jan. 26-29, 1998).

Metal shapes can be formed by physical vapor deposition of appropriatemetal layers on solid forms, which can be made of silicon using thetechniques described above, or which can be formed using other standardmold techniques such as embossing or injection molding. The metals areselectively removed using electropolishing techniques, in which anapplied anodic potential in an electrolytic solution will causedissolution of metals due to concentration of electric field lines. Oncethe underlying silicon forms have been exposed, the silicon isselectively etched away to form structures. This process could also beused to make structures made from other materials by depositing amaterial other than metal on the needle forms and following theprocedure described above.

Molds formed of silicon dioxide can be made by oxidizing the surface ofthe silicon mold forms, rather than depositing a metal and then etchingaway the solid needle forms to leave the hollow silicon dioxidestructures. In one embodiment, hollow, porous, or solid molds areprovided with longitudinal grooves or other modifications to theexterior surface of the molds.

Polymeric molds can be made using microfabricated molds. For example,the epoxy molds can be made as described above and injection moldingtechniques can be applied to form the structures. Thesemicromicromolding techniques are relatively less expensive to replicatethan the other methods described herein.

Solid Free Form Methods for Configuring the Molds

3DP is described by Sachs, et al., “CAD-Casting: Direct Fabrication ofCeramic Shells and Cores by Three Dimensional Printing” ManufacturingReview 5(2), 117-126 (1992) and U.S. Pat. No. 5,204,055 to Sachs, et al.3DP is used to create a solid object by ink-jet printing a binder intoselected areas of sequentially deposited layers of powder. Each layer iscreated by spreading a thin layer of powder over the surface of a powderbed. The powder bed is supported by a piston which descends upon powderspreading and printing of each layer (or, conversely, the ink jets andspreader are raised after printing of each layer and the bed remainsstationary). Instructions for each layer are derived directly from acomputer-aided design (CAD) representation of the component. The area tobe printed is obtained by computing the area of intersection between thedesired plane and the CAD representation of the object. The individualsliced segments or layers are joined to form the three dimensionalstructure. The unbound powder supports temporarily unconnected portionsof the component as the structure is built but is removed aftercompletion of printing.

SFF methods other than 3DP that can be utilized to some degree asdescribed herein are stereo-lithography (SLA), selective laser sintering(SLS), ballistic particle manufacturing (BPM), and fusion depositionmodeling (FDM). SLA is based on the use of a focused ultra-violet (UV)laser which is vector scanned over the top of a bath of aphotopolymerizable liquid polymer material. The UV laser causes the bathto polymerize where the laser beam strikes the surface of the bath,resulting in the creation of a first solid plastic layer at and justbelow the surface. The solid layer is then lowered into the bath and thelaser generated polymerization process is repeated for the generation ofthe next layer, and so on, until a plurality of superimposed layersforming the desired device is obtained. The most recently created layerin each case is always lowered to a position for the creation of thenext layer slightly below the surface of the liquid bath. A system forstereolithography is made and sold by 3D Systems, Inc., of Valencia,Calif., which is readily adaptable for use with biocompatible polymericmaterials. SLS also uses a focused laser beam, but to sinter areas of aloosely compacted plastic powder, the powder being applied layer bylayer. In this method, a thin layer of powder is spread evenly onto aflat surface with a roller mechanism. The powder is then raster-scannedwith a high-power laser beam. The powder material that is struck by thelaser beam is fused, while the other areas of powder remain dissociated.Successive layers of powder are deposited and raster-scanned, one on topof another, until an entire part is complete. Each layer is sintereddeeply enough to bond it to the preceding layer. A suitable systemadaptable for use in making medical devices is available from DTMCorporation of Austin, Tex.

BPM uses an ink-jet printing apparatus wherein an ink-jet stream ofliquid polymer or polymer composite material is used to createthree-dimensional objects under computer control, similar to the way anink-jet printer produces two-dimensional graphic printing. The device isformed by printing successive cross-sections, one layer after another,to a target using a cold welding or rapid solidification technique,which causes bonding between the particles and the successive layers.This approach as applied to metal or metal composites has been proposedby Automated Dynamic Corporation of Troy, N.Y. FDM employs an x-yplotter with a z motion to position an extrudable filament formed of apolymeric material, rendered fluid by heat or the presence of a solvent.A suitable system is available from Stratasys, Incorporated ofMinneapolis, Minn.

Cells for Forming Vascularized Tissue Layers

Although described herein with particular reference to formation ofvascularized tissue, it should be understood that the channels can beused for form lumens for passage of a variety of different fluids, notjust blood, but also bile, lymph, urine, and other body fluids, and forthe guided regeneration or growth of other types of cells, especiallynerve cells. The tissue layer may include some lumens for formingvasculature and some for other purposes, or be for one purpose,typically providing a blood supply to carry oxygen and nutrients to andfrom the cells in the tissue.

The tissue will typically include one or more types of “functional” orparenchymal cells, such as cells having specific metabolic functions,like hepatocytes, pancreatic cells, kidney, brain, reproductive tissuecells, cells forming intestine, nerve cells, bone, muscle, heart, skincells, etc. The vasculature will typically be formed from endothelialcells.

Cells can be obtained by biopsy or harvest from a living donor, cellculture, or autopsy. Cells can be dissociated using standard techniquessuch as digestion with collagenase, then seeded immediately into themold or after being maintained in cell culture. Cells can be normalcells or genetically engineered, from the patient into which the tissueis to be implanted, or from a suitable donor.

Methods for Seeding Cells into Molds

A method and materials to create complex vascularized living tissue inthree dimensions from a two-dimension microfabricated mold has beendeveloped. The method involved creating a two dimensional surface havinga branching structure etched into the surface. As shown in FIG. 2 a, ina preferred embodiment, the pattern in the mold 10 formed of a siliconwafer 11 begins with one or more large channels 12 which serially branchinto a large array of channels as small as individual capillaries 14 a,14 b, 14 c, etc., then converge to one or more large channels 16. Thecross section of the single “arterial” channel 12 and “venous” channel16 are shown in FIG. 2 b. The cross-sections of the portion of mold 10containing the “capillary” channels 14 a, 14 b, 14 c, etc., is shown inFIG. 2 c. The mold is shown in cross-section in FIG. 2 d, with a depthof approximately five microns.

The etched surface serves as a template within a mold formed with theetched surface for the circulation of an individual tissue or organ. Asshown in FIG. 4A, the mold pieces 30 and 32 are fitted together to makean enclosure 34, and the cells cultured. The vascular cells formvascular channels 36 based on the pattern etched in the mold, as shownin FIG. 4B. Once formed and sustained by their own matrix, the top 32 ofthe mold is removed, and the organ or tissue specific cells are thenadded to the etched surface, where they attach and proliferate to form athin, vascularized sheet of tissue 36. As shown in FIG. 4C, the tissuecan then be gently lifted from the mold using techniques such as fluidflow and other supporting material, as necessary.

Construction of Tissue or Organ Equivalents

After formation of the tissue layers, the tissue can be systematicallyfolded and compacted into a three-dimensional vascularized structure, asshown in FIG. 5. The two-dimensional surface of the mold can be variedto aid in the folding and compacting process. For example, the surfacecan be changed from planar to folded accordian like. It can be stackedinto multiple coverging plates. It could be curvilinear or have multipleprojections.

FIGS. 3A-G are perspective views of ways in which a single tissue layer36 (FIG. 3A) can be folded (FIGS. 3B and 3C) or stacked (FIG. 3D), orexpanded to form a balloon shape (FIG. 3E), funnel (FIG. 3F), or largelumen (FIG. 3G).

This structure can then be implanted into animals or patients bydirectly connecting the blood vessels to flow into and out of thedevice, as depicted in FIG. 6. Immediate perfusion of oxygenated bloodoccurs, which allows survival and function of the entire living mass.

Different types of tissue, or multiple layers of the same type oftissue, can be placed adjacent to each other prior to folding andcompacting, to create more complex or larger structures. For example, atubular system can be layered onto a vascular system to fabricateglomerular tissue and collecting tubules for kidneys. Bile duct tubescan be onlaid over vascularized liver or hepatocyte tissue, to generatea bile duct drainage system. Alveolar or airway tissue can be placed onlung capillaries to make new lung tissue. Nerves or lymphatics can beadded using variations of these same general techniques. Thetwo-dimensional surface of the mold can also be varied to aid in thefolding and compacting process. For example, the surface can be changedfrom planar to folded accordian like. It can be stacked into multiplecoverging plates. It could be curvilinear or have multiple projections.

EXAMPLE 1

Micromachining of Template to Tissue Engineer Branched VascularizedChannels for Liver Fabrication.

Micromachining technologies were used on silicon and pyrex surfaces togenerate complete vascular systems that may be integrated withengineered tissue before implantation. Trench patterns reminiscent ofbranched architecture of vascular and capillary networks were etchedusing standard photolithographic techniques onto silicon and pyrexsurfaces to serve as templates. Hepatocytes and endothelial cells werecultured and subsequently lifted as single-cell monolayers from thesetwo dimensional molds. Both cell types were viable and proliferative onthese surfaces. In addition, hepatocytes maintained albumin production.The lifted monolayers were then folded into compact three-dimensionaltissues. The goal is to lift these branched vascular networks from twodimensional templates so that they can be combined with layers ofparenchymal tissue, such as hepatocytes, to form three dimensionalconformations of living vascularized tissue for implantation.

Materials and Methods

Micromachining Techniques

Templates for the formation of sheets of living vascularized tissue werefabricated utilizing micromachining technology. For the present work, asingle level etch was utilized to transfer a vascular network patterninto an array of connected trenches in the surface of both silicon andpyrex wafers.

In this prototype, a simple geometry was selected for patterning thevascular network. Near the edge of each wafer, a single inlet or outletwas positioned, with a width of 500 μm. After a short length, the inletand outlet branched into three smaller channels of width 250 μm; each ofthese branched again into three 125 μm channels, and finally down tothree 50 μm channels. Channels extend from the 50 μm channels to form acapillary network, which comprises the bulk of the layout. In betweenthese inlet and outlet networks lies a tiled pattern of diamonds andhexagons forming a capillary bed and filling the entire space betweenthe inlet and outlet. In one configuration, the capillary width was setat 25 μm, while in the other, capillaries were fixed at 10 μm. Thisgeometry was selected because of its simplicity as well as its roughapproximation to the size scales of the branching architecture of theliver. Layout of this network was accomplished using CADENCE software(Cadence, Chelmsford, Mass.) on a Silicon Graphics workstation. A filewith the layout was generated and sent electronically to Align-Rite(Burbank, Calif.), where glass plates with electron-beam-generatedpatterns replicating the layout geometry were produced and returned forlithographic processing.

Starting materials for tissue engineering template fabrication werestandard semiconductor grade silicon wafers (Virginia Semiconductor,Powhatan, Va.), and standard pyrex wafers (Bullen Ultrasonics, Eaton,Ohio) suitable for MEMS processing. Silicon wafers were 100 mm diameterand 525 microns thick, with primary and secondary flats cut into thewafers to signal crystal orientation. Crystal orientation was <100>, andwafers were doped with boron to a resistivity of approximately 5 W-cm.The front surface was polished to an optical finish and the back surfaceground to a matte finish. Pyrex wafers were of composition identical toCorning 7740 (Corning Glass Works, Corning N.Y.), and were also 100 mmin diameter, but had a thickness of 775 microns. Both front and backsurfaces were polished to an optical finish. Prior to micromachining,both wafer types were cleaned in a mixture of 1 part H₂SO₄ to 1 partH₂O₂ for 20 minutes at 140° C., rinsed 8 times in deionized water with aresistivity of 18 MW, and dried in a stream of hot N₂ gas.

For silicon and pyrex wafers, standard photolithography was employed asthe etch mask for trench formation. Etching of pyrex wafers requiresdeposition of an intermediate layer for pattern transfer which isimpervious to the etch chemistry. A layer of polysilicon of thickness0.65 μm over the pyrex was utilized for this purpose. This layer wasdeposited using Low Pressure Chemical Vapor Deposition (LPCVD) at 570°C. and 500 mTorr via the standard silane decomposition method. In thecase of silicon, photoresist alone could withstand limited exposure totwo of the three etch chemistries employed. For the third chemistry, a1.0 μm layer of silicon dioxide was thermally deposited at 1100° C. inhydrogen and oxygen.

Once the wafers were cleaned and prepared for processing, images of theprototype branching architecture were translated onto the wafer surfacesusing standard MEMS lithographic techniques. A single layer ofphotoresist (Shipley 1822, MicroChem Corp., Newton, Mass.) was spun ontothe wafer surfaces at 4000 rpm, providing a film thickness ofapproximately 2.4 μm. After baking at 90° C. for 30 minutes, the layerof photoresist was exposed to uv light using a Karl Suss MA6 (SussAmerica, Waterbury, Vt.) mask aligner. Light was passed through thelithographic plate described earlier, which was in physical contact withthe coated wafer. This method replicates the pattern on the plate to anaccuracy of 0.1 μm. Following exposure, wafers were developed in Shipley319 Developer (MicroChem Corp., Newton, Mass.), and rinsed and dried indeionized water. Finally, wafers were baked at 110° C. for 30 minutes toharden the resist, and exposed to an oxygen plasma with 80 Watts ofpower for 42 seconds to remove traces of resist from open areas.

Silicon wafers were etched using three different chemistries, whilepyrex wafers were processed using only one technique. For pyrex, thelithographic pattern applied to the polysilicon intermediate layer wastransferred using a brief (approximately 1 minute) exposure to SF₆ in areactive-ion-etching plasma system (Surface Technology Systems, Newport,United Kingdom). Photoresist was removed, and the pattern imprinted intothe polysilicon layer was transferred into trenches in the silicon usinga mixture of 2 parts HNO₃ to 1 part HF at room temperature. With an etchrate of 1.7 microns per minute, 20 micron deep trenches were etched intothe pyrex wafers in approximately 12 minutes. Since the chemistry isisotropic, as the trenches are etched they become wider. Processing withthe layout pattern with 25 μm wide capillary trenches tended to resultin merging of the channels, while the use of 10 μm wide trenches avoidedthis phenomenon. Interferometric analysis of the channels after etchingshowed that surface roughness was less than 0.25 μm. Once channeletching of pyrex wafers was completed, polysilicon was removed with amixture of 10 parts HNO₃ to 1 part HF at room temperature, and waferswere re-cleaned in 1 part H₂SO₄ to 1 part HF.

Three different chemistries were employed to etch silicon in order toinvestigate the interaction between channel geometry and cell behavior.First, a standard anisotropic plasma etch chemistry, using a mixture ofSF₆ and C4F₈ in a switched process plasma system from STS²⁴, was used toproduce rectangular trenches in silicon. Narrower trenches are shallowerthan deep trenches due to a phenomenon known as RIE lag. A secondprocess utilized a different plasma system from STS, which producesisotropic trenches with a U-shaped profile. While the process isisotropic, widening of the trenches is not as severe as is experiencedin the isotropic pyrex etching process described earlier. In both ofthese plasma etching cases, trenches were etched to a nominal depth of20 μm. For the third process, anisotropic etching in KOH (45% w/w in H₂Oat 88° C.), the intermediate silicon dioxide layer mentioned above wasemployed. First, the silicon dioxide layer was patterned using HFetching at room temperature. The KOH process produces angled sidewallsrather than the rectangular profile or U-shaped profile produced by thefirst two recipes, respectively. Crystal planes in the <111> orientationare revealed along the angled sidewalls, due to anisotropic propertiesof the KOH etch process as a function of crystal orientation. Due to theself-limiting nature of the channels produced by this process, trenchdepth was limited to 10 μm. After completion of the silicon waferetching, all layers of photoresist and silicon dioxide were removed, andwafers were cleaned in 1 part H₂SO₄:1 part H₂O₂ at 140° C., followed byrinsing in deionized water and drying in nitrogen gas.

For this set of experiments, no attempt was made to alter the surfacechemistry of the silicon and pyrex wafers. Prior to processing, siliconwafers were uniformly hydrophobic, while pyrex wafers were equallyhydrophilic, as determined by observations of liquid sheeting andsessile drop formation. After processing, unetched surfaces appeared toretain these characteristics, but the surface chemistry within thechannels was not determined.

Animals

Adult male Lewis rats (Charles River Laboratories, Wilmington, Mass.),weighing 150-200 g, were used as cell donors. Animals were housed in theAnimal Facility of Massachusetts General Hospital in accordance with NIHguide lines for the care of laboratory animals. They were allowed ratchow and water ad libitum and maintained in 12-hour light and darkcycle.

Cell Isolations

Male Lewis rats were used as hepatic cell donors. HCs were isolatedusing a modification of the two-step collagenase perfusion procedure aspreviously by Aiken et al., J Pediatr Surg 25, 140 (1990); Seglen POMethods Cell Biol 13, 29 (1976). Briefly, the animals were anesthetizedwith Nembutal Sodium Solution (Abbott Laboratories, North Chicago,Ill.), 50 mg/kg, and the abdomen was prepared in sterile fashion. Amidline abdominal incision was made and the infrahepatic inferior venacava was cannulated with a 16-gauge angiocatheter (Becton Dickinson).The portal vein was incised to allow retrograde efflux and thesuprahepatic inferior vena cava was ligated. The perfusion was performedat a flow rate of 29 ml/min initially with a calcium-free buffersolution for 5 to 6 minutes, then with a buffer containing collagenasetype 2 (Worthington Biomedical Corp., Freehold, N.J.) at 37° C. Theliver was excised after adequate digestion of the extracellular matrixand mechanically agitated in William's E medium (Sigma, St. Louis, Mo.)with supplements to produce a single cell suspension. The suspension wasfiltered through a 300 micron mesh and separated into two fractions bycentrifugation at 50 g for 2 minutes at 4° C. The pellet containing theviable HC fraction was resuspended in William's E medium and furtherpurified by an isodensity Percoll centrifugation. The resulting pelletwas then resuspended in Hepatocyte Growth Medium, and cell counts andviabilities of HCs were determined using the trypan blue exclusion test.

The endothelial cells were derived from rat lung microvessels and theywere purchased directly from the vendor, Vascular Endothelial CellTechnologies (Rensellaer, N.Y.).

Hepatocyte Culture Medium

William's E medium supplemented with 1 g sodium pyruvate (Sigma, St.Louis, Mo.) and 1% glutamine-penicillin-streptomycin (Gibco BRL,Gaithersburg, Md.) were used during the cell isolation process. Theplating medium was Dulbecco's modified eagle medium (Gibco BRL)supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 44mM sodium-bicarbonate, 20 mM HEPES, 10 mM niacinamide, 30 microgram/mlL-proline, 1 mM ascorbic acid 2 phosphate, 0.1 microM dexamethasone(Sigma), insulin-transferrin-sodium selenite (5 mg/L-5 mg/L-5microgram/L, Roche Molecular Biomedicals, Indianapolis, Ind.), and 20ng/ml epidermal growth factor (Collaborative Biomedical Products,Bedford, Mass.).

Endothelial Cell Culture Medium

Dulbecco's modified eagle medium (Gibco BRL) was supplemented with 10%fetal bovine serum, 1% penicillin-streptomycin, 25 mg of ascorbic acid(Sigma), 10 mg L-alanine (Sigma), 25 mg L-proline (Sigma), 1.5 microgramcupric sulfate (Sigma), glycine (Sigma) and 1M Hepes buffer solution(Gibco BRL). The media was supplemented with 8 mg of ascorbic acid everyday.

Cell Attachment and Lifting from Non-Etched Silicon and Pyrex Wafers

Silicon and pyrex were both tested as possible substrates for theculture and lifting of endothelial cells and hepatocytes. Prior to cellseeding, the pyrex wafers were sterilized with 70% ethanol (Fisher,Pittsburg, Pa.) overnight and washed three times with sterile phosphatebuffered saline (Gibco BRL). Silicon wafers were first soaked in acetonefor1 hr, followed a methanol rinse for 15 minutes, and overnightsterilization in 100% isopropyl alcohol. Rat lung microvascularendothelial cells was cultured on non-coated pyrex and silicon surfaces,as well as wafers coated with vitrogen (30 microgramn/ml), Matrigel®(1%), or Gelatin (10 mg/ml). Once isolated, the cells were resuspendedin endothelial cell culture medium, seeded uniformly onto the wafer at adensity of 26.7×10³ cells/cm², and cultured at 5% CO₂ and 37° C. Afterreaching confluence, the ability of the monolayer of endothelial cellsto lift from the wafers was tested using a cell scrapper to promotedetachment.

The rat hepatocytes were also cultured on non-coated pyrex and silicon,as well as wafers coated with a thin and thick layers of vitrogen (30microgram/ml and 3 microgram/ml) and Matrigel (1%) in order to determinethe optimal methods for lifting hepatocyte sheets. Once isolated, thehepatocytes were resuspended in hepatocyte growth media, seeded onto thewafer at a density of 111.3×10³ cells/cm², and cultured at 5% CO₂ and37° C. Cell attachment and growth was observed daily using microscopyand cell lifting occurred spontaneously.

After determining which method for culturing was best for lifting thehepatocytes and endothelial cells in an intact layer, both membraneswere fixed in 10% buffered formalin for 1 hr and harvested forhistological study, and the hepatocytes were stainedimmunohistochemically.

Immunohistochemical Staining

The hepatocyte cell monolayer membrane was fixed in 10% bufferedformalin and processed for hematoxylin-eosin and immunohistochemicalstaining using a labeled streptavidin biotin method (LSAB2 kit for ratspecimen, DAKO, Carpinteria, Calif.). The primary antibody was rabbitanti-albumin (ICN, Costa Mesa, Calif.). Three-micron sections wereprepared and deparafinized. The specimens were treated with peroxidaseblocking buffer (DAKO) to prevent the nonspecific staining. Sectionswere stained with albumin diluted with phosphate buffered saline,followed by biotinylated anti-rabbit antibody and HRP conjugatedstreptavidin. Sections were treated with DAB as substrate and werecounterstained with hematoxylin.

Albumin Production

To assess hepatocyte function, albumin concentration in the culturemedium was measured every 24 hours for 5 days pre-cell detachment usingan enzyme linked immunosorbent assay (n=5), as described by Schwereetal., Clinica Chemica Acta 163, 237 (1987). In brief, a 96 wellmicroplate was coated with anti-rat albumin antibody (ICN). Afterblocking non-specific responses with a 1% gelatin solution, each samplewas seeded onto the plate and incubated for 1 hour. This was followed byanother 1 hour incubation with peroxidase conjugated anti-rat albuminantibody (ICN). Finally, the substrate was added and extinction wasmeasured with a microplate reader at 410 nm. R² of the standard curvewas >0.99.

Statistical Analysis

All data was expressed as mean ±SD. Statistical analysis was performedwith a paired t-test. Statistical significance was determined as whenthe p value of each test was less than 0.05.

Cell attachment to Etched Silicon and Pyrex Wafers

Endothelial cells and hepatocytes were also seeded onto etched siliconand pyrex wafers. Prior to cell seeding, the pyrex wafers weresterilized with 70% ethanol (Fisher) overnight and washed three timeswith sterile phosphate buffered saline (Gibco BRL). Silicon wafers werefirst soaked in acetone for 1 hr, followed a methanol rinse for 15minutes, and overnight sterilization in 100% isopropyl alcohol. Ontothese wafers were seeded rat lung microvascular endothelial cells at adensity of 26.7×10³ cells/cm², or rat hepatocytes at a density of111.3×10³ cells/cm². These cells were cultured at 5% CO₂ and 37° C., andtheir attachment and growth observed daily using microscopy.

Implantation of Hepatocyte Sheets into the Rat Omentum

Hepatocytes were cultured on silicon wafers coated with a thin layer ofvitrogen (30 microgram/ml), and lifted in sheets. Retrorsine is a drugknown to inhibit the regeneration of the normal liver by producing ablock in the hepatocyte cell cycle with an accumulation of cells in lateS and/or G₂ phase (Peterson JE J Pathol Bacteriol 89, 153 (1965)). Thisdrug was administered into the peritoneal cavity of two rats at a doseof 3 mg/ml/100 g on day 0, and after two weeks. Three weeks later, aportacaval shunt was created, and the following week a hepatocyte sheet,lifted after four days culture on vitrogen coated silicon (30microgram/ml), was implanted onto the microvasculature of the ratomentum and rolled into a three-dimensional cylinder, and a 60%hepatectomy was performed. The rolled omentum with hepatocytes washarvested at four weeks and at three months after implantation andanalyzed using histology.

Results

Micromachining

A schematic of the vascular branching network design used as a templatefor micromachining is shown in FIG. 9 a. This pattern was transferred tosilicon and pyrex wafers using the processes described in the Materialsand Methods section. Typical trench depths of 20 microns on silicon and10 microns on glass were achieved utilizing these processes. An opticalmicrograph of a portion of the capillary network etched into a siliconwafer is shown in FIG. 9 b. In FIG. 9 c, a Scanning Electron Micrographcross-section of an angled trench etched using the anisotropic etchingprocess described earlier is shown. This process resulted in excellentadhesion and enhanced lifting of living tissue.

Growth and Lifting of Cells from the Silicon and Pyrex Wafers

The adhesion and growth of endothelial cells and hepatocytes on severaldifferent substrate surfaces was compared. On all pyrex wafers, coatedor non-coated, the endothelial cells proliferated and grew to confluencewithin four days. These cells did not lift spontaneously, and whenscraped, did not lift as a single sheet. In addition, when thenon-coated silicon wafers were seeded with endothelial cells, the cellsheet fragmented upon lifting. On the other hand, endothelial cellsseeded onto silicon surfaces coated with vitrogen (30 microgram/ml),Matrigel (1%), and gelatin (10 mg/ml) did lift with the use ofmechanical means (i.e. a cell scraper), and provided an intact monolayersheet of endothelial cells. Upon observation, there were no significantdifferences in the effects of the three coatings on the detached cellsheets:

Hepatocytes also attached and spread well on all coated and non-coatedpyrex wafers, and did not lift spontaneously or in sheets when scrapedafter several days of growth. However, when seeded onto silicon wafers,they lifted spontaneously on all the non-coated and coated wafers. Thehepatocyte sheets lifted from the non-coated wafers after 3 days, butwere very fragile and fragmented easily. The monolayers that lifted fromthe thin and thickly coated vitrogen substrates (30 microgram/ml and 3microgram/ml) lifted after 4 days in culture to form an intacthepatocyte layer. Cells lifted from the Matrigel (1%) coated siliconwafers after 5 days in culture. There were no significant differences inappearance between the cell sheets lifted from the vitrogen and Matrigelcoated wafers.

Histological assessment of the detached cell monolayers of bothhepatocytes and endothelial cells manifested promising results.Hemotoxylin and Eosin (H&E) staining of both showed that all cells wereviable and that most were undergoing mitoses. The endothelial cells wereobserved to be primarily attenuated and to form a single-celledalignment. The monolayer of hepatocytes showed each cell to be of aspheriod configuration with eosinophilic floculent cytoplasm and a largenucleus with a bright red nucleolus, similar to that seen in the nativeliver. Moreover, cellular attachments were less attenuated than theendothelial cells. Thus, these results are reminiscent of each of thecell types' specific functions. In biological systems, the endotheliumfunctions to provide a thin, smooth outer surface of a barrier and atransport channel and so it is understandable that these cells areobserved here to be primarily attenuated and in a single-celled array.The hepatocytes have more of a tendency to form tissue and so less of asingle-celled array and more of a rounded multi-layered array is seen.

Albumin secretion into the hepatocyte culture medium at day 2, 3, 4, and5 was 165.96±29.87, 164.44±17.22, 154.33±18.46, 115.47±18.09(microgram/day, Graph 1), respectively. Though there was a statisticallysignificant difference between day 4 and day 5, no significantdifferences were observed between day 2, day 3, and day 4 (p<0.05 by thepaired t-test). Hence, this data shows that cells cultured on siliconwafers were able to maintain a fairly constant albumin production rateuntil day 4.

Moreover, through immunohistochemical staining of the detachedhepatocyte monolayers, many cells were stained positive for albuminindicating further that hepatocyte function was maintained on siliconwafers.

Implantation of Hepatocyte Sheet into the Rat Omentum

H&E staining of hepatocyte sheets implanted into rat omentumdemonstrated that all cells were viable and showed proliferation at fourweeks and three months. The implanted hepatocyte monolayer sheets, whenharvested, were over 5 cell layers thick in most areas.

This study demonstrates that silicon microfabrication technology can beutilized to form large sheets of living tissue. It also demonstrates thefeasibility of etching ordered branching arrays of channels that allowliving endothelial cells to line the luminal surface of the channels. Inaddition, it has been shown that organized sheets of engineeredhepatocyte tissue and endothelial tissue can be lifted from the surfaceof silicon or pyrex wafers and can be folded into a compact threedimensional configuration. The hepatocyte sheets have then been placedinto rats on the highly vascular surface of the omentum. That structurehas then been rolled into a three dimensional cylinder as a model for anengineered vasculature. Vascularized hepatic tissue was formed as apermanent graft.

Modifications and variations of the method and devices described hereinwill be obvious to those skilled in the art and are intended to beencompassed by the following claims.

1. A system for creating thin tissue layers comprising: (a) a moldcomprising a support surface having a pattern of channels thereon,suitable for attachment and culturing of cells to form lumens within thechannels, and suitable for attachment and culturing of a different typeof cells in the areas on the surface surrounding the channels.
 2. Themold of claim 1 wherein the system further comprises (b) connections andmeans for circulation of culture fluid through the mold for culturing ofthe cells attached thereto.
 3. The system of claim 1 wherein the moldhas branched channels therein, beginning from one or more inlets,expanding into more channels, and then converging back into one or moreoutlets.
 4. The system of claim 1 further comprising pumping means forcirculating fluid through the mold.
 5. The system of claim 1 wherein themold is formed from a material selected from the group consisting ofsilicon, glass, natural cell substrates like hydroxyapatite, andpolymer.
 6. The system of claim 1 further comprising a coating on thechannels which promotes adhesion and lifting of cells as intact sheetsfrom the mold.
 7. A method for making a mold comprising a supportsurface having a pattern of channels thereon, suitable for attachmentand culturing of cells to form lumens within the channels, and suitablefor attachment and culturing of a different type of cells in the areason the surface surrounding the channels, comprising: (a) selecting amaterial for forming the support surface from the group consisting ofsilicon, metals, polymers, and natural cell substrates likehydroxyapatite, (b) patterning channels in the surface of the materialto create lumens which can be seeded with cells to form tubularstructures for fluid flow, and (c) forming the mold so that cells can beseeded within the mold to form a thin layer of tissue surrounding thetubular structures.
 8. The method of claim 7 wherein the patterning isdone by a process selected from the group consisting of micromachining,lithography, etching, or molding.
 9. A method for forming complextissues comprising (a) selecting a mold having a complex pattern ofchannels formed into at least one surface into which cells can beseeded; (b) seeding cells into the channels to form blood vessels orother lumens; (c) culturing the cells under conditions until they formvessels or lumens; (d) seeding other type(s) of cells onto or into themold so that a tissue is formed incorporating the vessels or lumens; and(e) removing the tissue layer.
 10. The method of claim 9 furthercomprising (f) assembling multiple layers of the tissue layers until thedesired complex structure is formed.
 11. The method of claim 10 furthercomprising (g) implanting into a body the complex structure andanastomizing the vessles with the blood supply or lumens in other organsor tissues within the body.
 12. The method of claim 11 furthercomprising applying to the channel a coating on the channels whichpromotes adhesion and lifting of cells as intact sheets from the mold.13. A complex structure comprising multiple layers of tissue andvasculature formed by a method comprising (a) selecting a mold having acomplex pattern of channels formed into at least one surface into whichcells can be seeded; (b) seeding cells into the channels to form bloodvessels or other lumens; (c) culturing the cells under conditions untilthey form vessels or lumens; (d) seeding other type(s) of cells onto orinto the mold so that a tissue is formed incorporating the vessels orlumens; and (e) removing the tissue layer.
 14. The structure of claim 13wherein the vasculature comprises endothelial cells.
 15. The structureof claim 14 wherein the other cells are selected from the groupconsisting of parenchymal cells, cells forming cartilage or bone, musclecells, and nerve cells.
 16. The structure of claim 15 wherein theparenchymal cells are derived from organs selected from the groupconsisting of heart, liver, pancreas, intestine, brain, kidney,reproductive tissues and lung.